Kinetic parameters and tissue distribution of 5-oxo-L-prolinase determinedby a fluorimetric assay

5-Oxo-L-prolinase (5-OPase) catalyses the hydrolysis of 5-oxo-L-proline to glutamate with concomitant stoichiometric cleavage of ATP to ADP, a reactio n which is known to be part of the gamma-glutamyl cycle-an interrelated ser ies of reactions involved in the synthesis and metabolism of glutathione. A s recent studies indicate, this cyclic pathway plays a crucial role in the regulation of amino acid transport. Apparently, the intermediate product 5- oxo-L-proline functions as a second messenger molecule that upregulates the activity of certain amino acid transport systems. Thus, the degradation of 5-oxo-L-proline by 5-OPase leads to the downregulation of this stimulus. I n this study, a new sensitive fluorimetric assay for 5-OPase activity was e stablished which is based on the derivatization of glutamate with o-phthald ialdehyde in the presence of thiols and subsequent separation of the produc ts by HPLC. The method is suitable for the screening of chromatography frac tions as well as for the determination of the kinetic parameters K-m and V- max of purified 5-OPase. Additionally, it can be used for the measurement o f enzyme activity in crude cell extracts and evaluation of tissue distribut ion. (C) 1999 Elsevier Science B.V. All rights reserved.